Therapies targeting virus-host interactions are seen as promising strategies for treating gallid alphaherpesvirus 1 (ILTV) infection. Our study revealed a biphasic activation of two MAPK cascade pathways, MEK/ERK and p38 MAPK, as a notably activated host molecular event in response to ILTV infection. It exhibits antiviral functions at different stages of infection. Initially, the MEK/ERK pathway is activated upon viral invasion, leading to a broad suppression of metabolic pathways crucial for ILTV replication, thereby inhibiting viral replication from the early stage of ILTV infection. As the viral replication progresses, the p38 MAPK pathway activates its downstream transcription factor, STAT1, further hindering viral replication. Interestingly, ILTV overcomes this biphasic antiviral barrier by hijacking host p38-AKT axis, which protects infected cells from the apoptosis induced by infection and establishes an intracellular equilibrium conducive to extensive ILTV replication. These insights could provide potential therapeutic targets for ILTV infection.

ABSTRACT The tumor suppressor p53, primarily functioning as a transcription factor, has exhibited antiviral capabilities against various viruses in chickens, including infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). Nevertheless, the existence of a universal antiviral mechanism employed by chicken p53 (chp53) against these viruses remains uncertain. This study conducted a comprehensive comparison of molecular networks involved in chp53’s antiviral function against IBDV, ALV-J, and ILTV. This was achieved through an integrated analysis of ChIP-seq data, examining chp53’s genome-wide chromatin occupancy, and RNA-seq data from chicken cells infected with these viruses. The consistent observation of chp53 target gene enrichment in metabolic pathways, confirmed via ChIP-qPCR, suggests a ubiquitous regulation of host cellular metabolism by chp53 across different viruses. Further genome binding motif conservation analysis and transcriptional co-factor prediction suggest conserved transcriptional regulation mechanism by which chp53 regulates host cellular metabolism during viral infection. These findings offer novel insights into the antiviral role of chp53 and propose that targeting the virus-host metabolic interaction through regulating p53 could serve as a universal strategy for antiviral therapies in chickens. IMPORTANCE The current study conducted a comprehensive analysis, comparing molecular networks underlying chp53’s antiviral role against infectious bursal disease virus (IBDV), avian leukosis virus subgroup J (ALV-J), and avian infectious laryngotracheitis virus (ILTV). This was achieved through a combined assessment of ChIP-seq and RNA-seq data obtained from infected chicken cells. Notably, enrichment of chp53 target genes in metabolic pathways was consistently observed across viral infections, indicating a universal role of chp53 in regulating cellular metabolism during diverse viral infections. These findings offer novel insights into the antiviral capabilities of chicken p53, laying a foundation for the potential development of broad-spectrum antiviral therapies in chickens.

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53–Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it.

Poultry farming is one of the pillar industries of global animal husbandry. In order to guarantee production, poultry are frequently vaccinated from the moment they are hatched. Even so, the initial immunity of chicks is still very poor as they are in the “window period” of immune protection. In ovo vaccination pushes the initial immunization time forward to the incubation period, thereby providing earlier immune protection for chicks. In ovo vaccination is currently a research hotspot of poultry disease prevention and control, which is in line with the intensification of poultry production. However, the vaccines currently available for in ovo vaccination are limited and cannot meet the needs of industrial development, so how to efficiently activate the adaptive immune response of chicken embryos becomes the key to restrict product development and technological progress of in ovo vaccination. Its breakthrough, to a large extent, depends on systematic illustration of the mechanism underlying the adaptive immune response post immunization. Clarification of this issue will provide us with theoretical support and potential solutions for the development of novel vaccines for in ovo vaccination, the augmentation of efficacy of current vaccines and the optimization of immune programs.

Treatment options for herpesvirus infections that target the interactions between the virus and the host have been identified as promising. Our previous studies have shown that transcription factors p53 and Fos are essential host determinants of gallid alpha herpesvirus 1 (ILTV) infection. The impact of p53 and Fos on ILTV replication has ‘not been fully understood yet. Using the sole ILTV-permissive chicken cell line LMH as a model, we examined the effects of hosts p53 and Fos on all phases of ILTV replication, including viral gene transcription, viral genome replication, and infectious virion generation. We achieved this by manipulating the expression of p53 and Fos in LMH cells. Our results demonstrate that the overexpression of either p53 or Fos can promote viral gene transcription at all stages of the temporal cascade of ILTV gene expression, viral genome replication, and infectious virion production, as assessed through absolute quantitative real-time PCR, ILTV-specific RT-qPCR assays, and TCID50 assays. These findings are consistent with our previous analyses of the effects of Fos and p53 knockdowns on virus production and also suggest that both p53 and Fos may be dispensable for ILTV replication. Based on the synergistic effect of regulating ILTV, we further found that there is an interaction between p53 and Fos. Interestingly, we found that p53 also has targeted sites upstream of ICP4, and these sites are very close to the Fos sites. In conclusion, our research offers an in-depth understanding of how hosts p53 and Fos affect ILTV replication. Understanding the processes by which p53 and Fos regulate ILTV infection will be improved by this knowledge, potentially paving the way for the development of novel therapeutics targeting virus–host interactions as a means of treating herpesvirus infections.

The tumor suppressor p53, which acts primarily as a transcription factor, can regulate infec-tions from various viruses in chickens. However, the underlying mechanisms of the antiviral functions of chicken p53 (chp53) remain unclear due to the lack of detailed information on its transcriptional regulation. Here, to gain comprehensive insights into chp53 tran-scriptional regulatory function in a global and unbiased manner, we determined the genome-wide chromatin occupancy of chp53 by chromatin immunoprecipitation, which was followed by sequencing and chp53-mediated gene expression profile by RNA sequencing using chemi-cally immortalized leghorn male hepatoma (LMH) cells with ectopic expression of chp53 as the model. The integrated parallel genome-wide chromatin occupancy and gene expression analysis characterized chp53 chro-matin occupancy and identified 754 direct target genes of chp53. Furthermore, functional annotation and cross -species comparative biological analyses revealed the con-served key biological functions and DNA binding motifs of p53 between chickens and humans, which may be due to the consensus amino acid sequence and structure of p53 DNA-binding domains. The present study, to our knowledge, provides the first comprehensive characteri-zation of the chp53 transcriptional regulatory network, and can possibly help to improve our understanding of p53 transcriptional regulatory mechanisms and their antiviral functions in chickens.

Therapeutics targeting virus-host interactions have been considered promising strategies for treating herpesvirus infection. Our previous study on avian infectious laryngotracheitis virus (ILTV), an avian herpesvirus economically important to the poultry industry worldwide, identified the small molecule Pifithrin-α (PFT-α) as a potential therapeutic agent. However, the underlying mechanisms of its antiviral function remain largely unknown. Using the ILTV-permissive chicken cell line LMH as the model, we found that PFT-α effectively suppressed the transcription and genome replication of ILTV and greatly reduced the level of infectious virions. Genome-wide transcriptome analysis revealed extensive repression of the metabolic processes of infected cells by PFT-α administration. Further metabolome assays of ILTV-infected cells using liquid chromatography coupled with mass spectrometry suggest host nucleotide metabolism and ATP synthesis as the key targets of PFT-α treatment during its repression of ILTV replication, which was experimentally supported by the reduced transcription of many key enzymes essential to nucleotide metabolism and ATP synthesis. The present study provides insights into the mechanisms by which PFT-α inhibits ILTV infection, which may increase the probability of successful clinical application of this molecule.

Avian Tembusu virus (TMUV) is a newly emerging avian pathogenic flavivirus that spreads rapidly, has an expanding host range and undergoes cross-species transmission. Our previous study identified avian monocytes/macrophages as the key targets of TMUV infection, since the infection of host monocytes/macrophages was crucial for the replication, transmission, and pathogenesis of TMUV. The polarization of host macrophages determines the functional phenotypes of macrophages; however, the effect of TMUV infection on macrophage polarization remains unclear. Here, we analysed the expression spectra of the marker genes of macrophage polarization upon TMUV infection in the HD11 chicken macrophage cell line and primary monocytes/macrophages isolated from the peripheral blood of specific pathogen-free (SPF) chickens and ducks. We found that viral replication mainly induced M1 marker genes and triggered nitric oxide (NO) release at different levels, suggesting that TMUV infection led mainly to host macrophages polarizing into the classically activated (M1) type. The NO that was increased upon infection did not function as an antiviral agent against TMUV, since the replication of TMUV in HD11 cells was not affected by the addition of an organic NO donor. Furthermore, upon TMUV infection, polarized HD11 cells exhibited increased migration but reduced phagocytosis, as evidenced by scratch assay and neutral red uptake assay, respectively. Our present study characterized the polarization of host monocytes/macrophages upon TMUV infection, which may lay a foundation for further research on the immune escape mechanism and pathogenic mechanism of TMUV.

Ducks are an economically important waterfowl but a natural reservoir for some zoonotic pathogens, such as influenza virus and flaviviruses. Our understanding of the duck immune system and its interaction with viruses remains incomplete. In this study, we constructed the transcriptomic landscape of duck circulating immune cells, the first line of defense in the arthropod-borne transmission of arboviruses, using high-throughput single-cell transcriptome sequencing, which defined 14 populations of peripheral blood leukocytes (PBLks) based on distinct molecular signatures and revealed differences in the clustering of PBLks between ducks and humans. Taking advantage of in vivo sex differences in the susceptibility of duck PBLks to avian tembusu virus (TMUV) infection, a mosquito-borne flavivirus newly emerged from ducks with a broad host range from mosquitos to mammals, a comprehensive comparison of the in vivo dynamics of duck PBLks upon TMUV infection between sexes was performed at the single-cell level. Using this in vivo model, we discovered that TMUV infection reprogrammed duck PBLks differently between sexes, driving the expansion of granulocytes and priming granulocytes and monocytes for antiviral immune activation in males but decreasing the antiviral immune activity of granulocytes and monocytes by restricting their dynamic transitions from steady states to antiviral states with a decrease in the abundance of circulating monocytes in females. This study provides insights into the initial immune responses of ducks to arthropod-borne flaviviral infection and provides a framework for studying duck antiviral immunity.

Gallid alpha-herpesvirus 1, also known as avian infectious laryngotracheitis virus (ILTV), continues to cause huge economic losses to the poultry industry worldwide. Similar to that of other herpesvirus-encoded proteins, the expression of viral genes encoded by ILTV is regulated by a cascade, and the underlying regulatory mechanism remains largely unclear. The viral immediate-early (IE) gene ICP4 plays a prominent role in the initiation of the transcription of early and late genes during ILTV replication. In this study, we identified AP-1 as the key regulator of the transcription of ILTV genes by bioinformatics analysis of genome-wide transcriptome data. Subsequent functional studies of the key members of the AP-1 family revealed that Fos, but not Jun, regulates ILTV infection through AP-1 since knockdown of Fos, but not Jun, by gene silencing significantly reduced ICP4 transcription and subsequent viral genome replication and virion production. Using several approaches, we identified ICP4 as a bona fide target gene of Fos that regulated Fos and has Fos response elements within its promoter. Neither the physical binding of Jun to the promoter of ICP4 nor the transcriptional activity of Jun was observed. In addition, knockdown of Fos reduced the transcription of MDH1 and ATP5A1, genes encoding two host rate-limiting enzymes essential for the production of the TCA intermediates OAA and ATP. The biological significance of the transcriptional regulation of MDH1 and ATP5A1 by Fos in ILTV infection was supported by the fact that anaplerosis of OAA and ATP rescued both ICP4 transcription and virion production in infected cells under when Fos was silenced. Our study identified the transcription factor Fos as a key regulator of ILTV infection through its transcription factor function on both the virus and host sides, improving the current understanding of both avian herpesvirus–host interactions and the roles of AP-1 in viral infection.

Although therapeutics targeting viral metabolic processes have been considered as promising strategies to treat herpesvirus infection, the metabolic requirements of gallid alphaherpesvirus 1 (ILTV), which is economically important to the poultry industry worldwide, remain largely unknown. Using the ILTV-susceptible but nonpermissive chicken cell line DF-1 and the ILTV-permissive chicken cell line LMH as models, the present study explored the metabolic requirements of ILTV by global transcriptome analysis and metabolome assays of ILTV infected cell lines in combination with a set of functional validations. The extensive metabolic exploration demonstrated that ILTV infection tended to promote a metabolic shift from glycolysis to fatty acid (FA) and nucleotide biosynthesis and utilizes glutamine independently of glutaminolysis, without significant general effect on the TCA cycle. In addition, different metabolic pathways were found to be required for distinct stages of ILTV replication. Glucose and glutamine were required for the transcription of viral immediate early gene ICP4 and subsequent steps of viral replication. However, FA synthesis was essential for assembly but not required for other upstream steps of ILTV replication. Moreover, the metabolic requirements of ILTV infection revealed in chicken cell lines were further validated in chicken primary cells isolated from chicken embryo kidneys and chicken embryo livers. The present study, to the best of our knowledge, provides the first global metabolic profile of animal herpesviruses and illustrates the main characteristics of the metabolic program of ILTV.

Viral spread is considered a promising target for antiviral therapeutics, but the associated mechanisms remain unclear for gallid alpha herpesvirus 1 (ILTV). We previously identified proto-oncogene tyrosine-protein kinase Src (Src) as a crucial host determinant of ILTV infection. The present study revealed accelerated spread of ILTV upon Src inhibition. This phenomenon was independent of either viral replication or the proliferation of infected cells and could not be compromised by neutralizing antibody. Neither extracellular vesicles nor the direct cytosol-to-cytosol connections between adjacent cells contributed to the enhanced spread of ILTV upon Src inhibition. Further genome-wide transcriptional profile analyses in combination with functional validation identified fatty acid metabolism as an essential molecular event during modulation of the intercellular spread and subsequent cytopathic effect of ILTV by Src. Overall, these data suggest that Src controls the cell-to-cell spread of ILTV in a cellular fatty acid metabolism-dependent manner, which determines the virus's cytopathic effect.

Mosquito-borne flaviviruses include a group of pathogenic viruses that cause serious diseases in humans and animals, including dengue, West Nile, and Japanese encephalitis viruses. These flaviviruses are zoonotic and use animals, including birds, as amplifying and reservoir hosts. Avian Tembusu virus (TMUV) is an emerging mosquito-borne flavivirus that is pathogenic for many avian species and can infect cells derived from mammals and humans in vitro . Although not currently pathogenic for primates, the infection of duck industry workers and the potential risk of TMUV infection in immunocompromised individuals have been highlighted. Thus, the prevention of TMUV in flocks is important for both avian and mammalian health. Our study reveals the escape of TMUV from the first line of the host defense system in the arthropod-borne transmission route of arboviruses, possibly helping to extend our understanding of flavivirus infection in birds and refine the design of anti-TMUV therapeutics.